The milkcap genus Lactifluus is one of the most common ectomycorrhizal genera within Central African rainforests. During a field trip to the Dja Biosphere Reserve in Cameroon, a new Lactifluus species was found. Molecular and morphological analyses indicate that the species belongs to Lactifluus section Xerampelini and we formally describe it here as Lactifluus bicapillus sp. nov.

Ectomycorrhizal fungi, Gilbertiodendron, Lactarius , phylogeny, taxonomy, tropical Africa, Uapaca

Rainforests occur in Central Africa and form the main vegetation type in the Guineo-Congolian region (White 1983). Large parts of southern Cameroon and northern Gabon are covered by rainforest, characterised by high humidity, closed canopies, and competition for light in the understory. Common tree species within these rainforests, such as the Dja Biosphere Reserve, include ectomycorrhizal (ECM) species from the Phyllanthaceae (e.g. Uapaca spp. Baill.) and the Fabaceae (i.e. Gilbertiodendron dewevrei (De Wild.) J.Léonard) (Sonké and Couvreur 2014). Uapaca species mainly occur mixed with other tree species, whereas G. dewevrei forms more or less monodominant stands, mixed with an occasional Uapaca species. These trees are typical hosts for ECM fungi and Russulaceae have been repeatedly recorded as associated with these trees (Verbeken and Walleyn 2010; De Crop et al. 2016; Delgat et al. 2017; T.W. Henkel pers. comm.).

Within Central African rainforests, the ECMRussulaceae genera Russula Pers. and Lactifluus (Pers.) Roussel are abundant (Douanla-Meli and Langer 2009; Verbeken and Buyck 2002; Verbeken et al. 2008; Verbeken and Walleyn 2010). The milkcap genus Lactifluus is mainly distributed in the tropics (De Crop et al. 2017). It is a species-rich genus with about 160 species distributed worldwide, of which the majority is found in tropical Asia (Le et al. 2007b; Stubbe et al. 2010; Van de Putte et al. 2010), tropical Africa (Van de Putte et al. 2009; Verbeken and Walleyn 2010; De Crop et al. 2012, 2016; Maba et al. 2014, 2015a, b; Delgat et al. 2017; De Lange et al. 2018) and the Neotropics (Henkel et al. 2000; Miller et al. 2002; Smith et al. 2011; Sá et al. 2013; Sá and Wartchow 2013). The genus is relatively understudied and many species remain undescribed due to this mainly tropical distribution. Furthermore, the genus is known for its many species complexes with morphologically cryptic species (Stubbe et al. 2010; Van de Putte et al. 2010, 2012; De Crop et al. 2014; De Crop et al. 2017).

About 20 Lactifluus species are known from the rainforests of Central Africa (Verbeken and Walleyn 2010). The actual diversity is expected to be higher for several reasons: (i) the ECM flora is present in most parts of the tropical African rainforest, (ii) most countries in the region are understudied due to difficult political situations or challenging sampling conditions, (iii) seasonality in the rainforest is less pronounced, which makes it difficult to assess the exact fruiting period of these fungi and the fruiting of fungi can be missed during short sampling periods, and (iv) Lactifluus is known for its morphologically cryptic diversity with several species complexes occurring. Traditional species descriptions were often based on morphology and this morphologically cryptic diversity makes it difficult to correctly assess the number of species based on morphology alone.

During fieldwork in Cameroon in 2012 and 2014, several Lactifluus specimens were found morphologically resembling yet different from the described species within L. subg. Pseudogymnocarpi (Pacioni & Lalli) De Crop. The phylogenetic results of De Crop et al. (2017), based on four nuclear genes, revealed that this species is new to science. A preliminary microscopic study confirmed the deviating morphology of the Cameroonian collections and a more detailed study of all available material was initiated. In this study, molecular and morphological examinations were performed, the collections were compared with closely related species, and a new species, Lactifluus bicapillus, was described based on these results.

Our molecular results show that the recently collected specimens form a well-supported monophyletic clade within Lactifluus subg. Pseudogymnocarpi, L. sect. Xerampelini (Fig. 1). The species is sister to a well-supported clade of all other species within this section, with L. xerampelinus (Karhula & Verbeken) Verbeken being its closest relative. Morphological and ecological data confirm that these collections are different from all other species in L. sect. Xerampelini, therefore the new species is described here as Lactifluus bicapillus sp. nov.

Figure 1. 

Overview Maximum Likelihood tree of Lactifluus subg. Pseudogymnocarpi, based on concatenated ITS and LSU sequence data. Sequences of the here described species Lactifluus bicapillus are written in bold. Maximum Likelihood bootstrap values > 70 are shown. Numbers of undescribed sections refer to De Crop et al. (2017).

Taxonomy

Lactifluus bicapillus Lescroart & De Crop

MycoBank No: 827400

Figs 2, 3, 4

Diagnosis

Lactifluus bicapillus differs from L. xerampelinus by its yellowish-orange to dark red cap, fertile lamella edge, a lampropalisade with two types of terminal elements as pileipellis type, and a distribution in the Guineo-Congolian rainforest.

Holotype

CAMEROON. East Region, Haut-Nyong division, Somalomo subdivision, Dja Biosphere Reserve, alt. ca 640 m, 3°21.83'N, 12°44.18'E, rainforest with Uapaca paludosa and U. guineensis, 14 May 2014, leg.: De Crop & Verbeken, EDC 14-249 (GENT!).

Basidiocarps medium-sized. Pileus 34–79 mm in diameter, firm, infundibuliform to deeply infundibuliform, planoconvex with central depression when younger; margin involute when juvenile, becoming inflexed up to reflexed when older; edge entire, sometimes eroded when older; surface felty to chamois leather-like, often slightly pruinose in the centre, often grooved, concentrically wrinkled, in young specimens completely velutinous and somewhat translucent; rubiginous (7D6–7) in centre, becoming paler and more orange towards the margin (6C5–6 to 5A5–6); young specimens dark reddish or burgundy in centre, to bright orange or yellow at the margin (8F6 to 7B6, to 6A5, 4AG); secondary velum absent. Stipe 16–39 × 6–12 mm, cylindrical to slightly tapering downwards, often laterally curved near the base, central to eccentric insertion to pileus, entire or bruised appearance, sometimes with white flocks near the base; surface smooth and felty, sometimes pruinose, yellowish orange (5AB5–6), becoming slightly paler and more yellow near the base and/or lamellae (5A4–5). Lamellae intervenose, transvenose, sparingly bifurcating; attachment adnate to decurrent with some lamellae forming a small tooth; juveniles not brittle, rather thin, older specimens brittle to very brittle, thick to very broad; edge entire and concolourous; distant, 3–5 + 6–9 L+l/cm, between 2 lamellae often 3 lamellulae, with regular short long-short pattern; creamy yellow (3A2) to yellowish orange (4A4). Context white, with a faint yellow tinge, colour not changing when cut, but in 1 collection (EDC 14-238) becoming brown when damaged, rather solid and full, smell sweet or not distinct, taste mild. Latex white, somewhat astringent, rather abundant, becomes less abundant and more watery with age, mild, colour rarely changing brownish when isolated. Chemical reactions unchanging with Fe₂SO₄; context faint blue after 5 sec. with guaiac.

Basidiospores [6.2]–7.3–7.9–[9.6](10.3) × [4.6]–5.5–5.9–[6.8] μm; ellipsoid, with Q = (1.22)1.31–1.39(1.51); ornamentation amyloid, composed of low ridges and warts, up to 0.2 μm high, forming an incomplete to complete reticulum; plage inamyloid or centrally amyloid. Basidia 43–62 × 8–12 μm, rather long, narrowly subclavate, 1-, 2- or 4-spored; content oleiferic. Sterile elements abundant, 19.5–40 × 3.5–5.5 μm, not emergent, cylindrical, septate with clamp-like bulges under the septum, with rounded apex. Pleurocystidia absent. Pleuropseudocystidia very scarce in mature specimens, abundant in young specimens, narrowly and irregular cylindrical to flexuose, 3.3–4.6 μm diam., not emerging, apex obtuse, oleiferic content. Lamellae-edge fertile, consisting of basidioles with some basidia. Marginal cells absent. Hymenophoral trama cellular, with sphaerocytes and abundant lactifers. Pileipellis a lampropalisade, up to 275 μm thick; terminal elements of two types, without transitional forms: the first type long and slender, thick-walled and often septate, with a wide base, up to 7 μm, and growing thinner towards the apex, down to 1–2 μm, length 52–92 μm, often narrowing rather abruptly, and twisted; the second type short and broad, also thick-walled and often septate, not specifically narrower towards the apex, often twisted, 20–44 × 5–7 μm; subpellis composed of mostly rounded cells. Stipitipellis similar to pileipellis but not as thick; terminal elements of the long type 52–75 × 5–7 μm; terminal elements of the short type 22–29 × 5–7 μm. Clamp-connections absent.

Figure 2. 

Basidiomata of Lactifluus bicapillus. a–c Basidiomata of Lactifluus bicapillus (EDC 12-176, EDC 12-174, holotype EDC 14-249 resp.) d Detail of lamellae (EDC 14-176), e) young specimen (EDC 12-169) f Detail of latex (EDC 12-169) g Detail of brown colour change of the latex (EDC 14-238) (photographs a–f by E. De Crop, g by A. Verbeken).

Figure 3. 

Microscopic features of Lactifluus bicapillus a Basidiocarps (from EDC 12-071, EDC 12-169, EDC 12-174, EDC 12-176, and EDC 14-249) b Basidia (from EDC 12-071, and EDC 14-249) c Sterile elements from the hymenium (from EDC 12-169) d Pleuropseudocystidia (from EDC 12-169) e Basidiospores (from EDC 14-249). Illustrations by E. De Crop, J. Lescroart and A. Verbeken. Scale bar: 10 μm.

Figure 4. 

Microscopic features of Lactifluus bicapillus (continued) a Terminal elements of the pileipellis (from EDC 12-071) b Terminal elements of the stipitipellis (from EDC 12-176) c Section through the pileipellis (from holotype EDC 14-249). Illustrations by E. De Crop and J. Lescroart. Scale bar: 10 μm.

Distribution

Known from Cameroon and Gabon.

Ecology

Guineo-Congolian rainforest, scattered on forest floor under Gilbertiodendron dewevrei, Uapaca guineensis, U. acuminata, and U. paludosa.

Etymology

A combination of ‘bi’ and ‘capillus’, referring to the two types of terminal elements in the pileipellis and stipitipellis.

Conservation status

Unknown.

Specimens examined

Cameroon. East Region, Haut-Nyong division, Somalomo subdivision, Koulou village, alt. ca 650 m, 3°23.61'N, 12°44.50'E, rainforest, Gilbertiodendron dewevrei, Uapaca guineensis, U. acuminata, 15 May 2012, De Crop, EDC 12-071 (GENT); East Region, Haut-Nyong division, Lomié subdivision, Bosquet village, alt. ca 610 m, 3°07.82'N, 13°53.36'E, rainforest with many Uapaca trees, on a riverbank, Uapaca guineensis, 24 May 2012, De Crop, EDC 12-169 (GENT); Ibidem, Gilbertiodendron dewevrei, De Crop, EDC 12-174 (GENT); Ibidem, Uapaca guineensis, EDC 12-176 (GENT); East Region, Haut-Nyong division, Somalomo subdivision, Dja Biosphere Reserve, alt. ca 650 m, 3°21.90'N, 12°44.15'E, rainforest, Uapaca paludosa, U. guineensis, 14 May 2014, De Crop & Verbeken, EDC 14-238 (GENT); Ibidem, alt. ca 640 m, 3°21.83'N, 12°44.18'E, De Crop & Verbeken, EDC 14-249 (GENT); Ibidem, alt. ca 650 m, 3°19.87'N, 12°45.42'E, rainforest, near the river, Uapaca sp., 17/05/2014, De Crop & Verbeken, EDC 14-284 (GENT).

Lactifluus bicapillus is recognized in the field by its yellowish-orange to dark-red cap, a concolourous or somewhat paler stipe, yellow lamellae, and unchanging white latex. L. bicapillus is placed in L. subg. Pseudogymnocarpi, L. sect. Xerampelini. Species in this section are characterized by yellowish-orange to reddish-brown caps, a palisade-like structure as pileipellis, the absence of true pleurocystidia, and generally low ornamented basidiospores (not higher than 0.2 μm) ranging from verrucose to almost completely reticulate (De Crop et al. 2017). Lactifluus bicapillus perfectly concurs with these morphological characteristics, providing additional support for its placement in L. sect. Xerampelini.

Lactifluus sect. Xerampelini is exclusively known from Africa and contains six described species (Fig. 5): L. goossensiae (Beeli) Verbeken, L. kivuensis (Verbeken) Verbeken, L. persicinus Delgat & De Crop, L. pseudovolemus (R. Heim) Verbeken, L. rubiginosus (Verbeken) Verbeken, and L. xerampelinus (Verbeken and Walleyn 2010; Delgat et al. 2017).

Figure 5. 

Basidiomata of described species of Lactifluus sect. Xerampelinia L. xerampelinus (EDC 11-113) bL.kivuensis (JR Z 233) cL.cf. pseudovolemus (ADK 2968) d L. rubiginosus (EDC 11-120) e L. persicinus (EDC 12-001, holotypus) f L. bicapillus (EDC 14-249, holotypus) (photographs a, d–f by E. De Crop, b by J. Rammeloo and c by A. De Kesel).

Lactifluus bicapillus differs in ecology from all but one species of L. sect. Xerampelini. Species from this section occur in woodlands, gallery forests and rainforests (Verbeken and Walleyn 2010). Lactifluus xerampelinus and L. rubiginosus are found in miombo woodland in East Africa, while L. goossensiae is known from both Sudanian woodland and Central African gallery forests. Lactifluus persicinus and L. pseudovolemus occur in West African gallery forests. Both L. kivuensis and L. bicapillus are found in the Guineo-Congolian rainforest, associated with Gilbertiodendron dewevrei and Uapaca species.

Macroscopically, L. bicapillus differs from the other species of this section by a combination of bright cap colours, which vary from dark red to bright orange near the edge, cream white lamellae and pale yellow-orange stipe colours in adult basidiocarps (Fig. 5).

All species from L. sect. Xerampelini have ellipsoid to elongate basidiospores, with amyloid ornamentation composed of very low warts and ridges (up to 0.2 µm high) that are isolated, aligned or forming an incomplete reticulum. All seven species have long and slender basidia, mostly cylindrical and 4-spored. However, 1- and 2-spored basidia are present in L. bicapillus, L. persicinus, and L. pseudovolemus. True cystidia are absent in all species. Pleuropseudocystidia are scarce in L. bicapillus, L. persicinus, and L. kivuensis, abundant in the other species. These pleuropseudocystidia are occasionally emergent in all species; however, emergent pleuropseudocystidia were not observed in L. bicapillus. Lactifluus persicinus and L. bicapillus have a fertile lamellar edge, whilst the others have a sterile lamellar edge (or unknown in L. pseudovolemus and L. goossensiae).

All species of this section have palisade-like structures as pileipellis. Lactifluus bicapillus, L. persicinus, and L. goossensiae have a lampropalisade with thick-walled terminal elements. Lactifluus pseudovolemus has a palisade in which the elements of the pileipellis are slightly thickened. Lactifluus kivuensis, L. xerampelinus, and L. rubiginosus have a palisade to trichopalisade, with only thin-walled elements of the pileipellis. Only Lactifluus bicapillus, L. persicinus, and L. goossensiae have terminal elements that are narrow near the apex. Furthermore, L. bicapillus is the only species within this section with two types of terminal elements in the pilei- and stipitipellis.

With the finding of Lactifluus bicapillus, L. sect. Xerampelini now contains seven described species, all from sub-Saharan Africa. Together with the recently described L. persicinus (Delgat et al. 2017), L. bicapillus was found during two sampling expeditions in Cameroon. Even though those expeditions only covered a small area of the Guineo-Congolian rainforest and gallery forests, we collected at least five species new to science (De Crop et al. 2017). This highlights the large Lactifluus diversity in Africa, with many areas still unexplored and probably many new species still to be found.

The first author is supported by the “Special Research Fund Ghent University” (BOF, grants B/13485/01 and BOF-PDO-2017-001201). The 2012 survey in Cameroon was financially supported by the Faculty Committee Scientific Research (FCWO) of Ghent University. The 2014 survey in Cameroon was financially supported by the Research Foundation Flanders (FWO, grant V416214N) and by the King Leopold III Fund for Nature Exploration and Conservation. We express our gratitude to all who helped during fieldwork, especially to the conservators and Ecogards in post in the Dja Biosphere Reserve (from 2012 to 2014) and Mr Tchana Tchonkui Merlin. We would like to thank Viki Vandomme for conducting lab work. We thank André De Kesel and Jan Rammeloo for providing pictures of Lactifluus species. We thank the reviewers and the editor for their constructive suggestions and detailed comments on the manuscript.